CN101973990B - Method for separating dehydrocorydaline by pH-zone-refining countercurrent chromatography - Google Patents
Method for separating dehydrocorydaline by pH-zone-refining countercurrent chromatography Download PDFInfo
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- dehydrocorydaline
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- chloroform
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- corydalis tuber
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- RFKQJTRWODZPHF-UHFFFAOYSA-N Dehydrocorydaline Chemical compound COC1=C(OC)C=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C(C)=C3C2=C1 RFKQJTRWODZPHF-UHFFFAOYSA-N 0.000 title claims abstract description 124
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000007670 refining Methods 0.000 title claims abstract description 13
- 238000004185 countercurrent chromatography Methods 0.000 title abstract description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 94
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 60
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 45
- 239000000287 crude extract Substances 0.000 claims abstract description 33
- 239000012488 sample solution Substances 0.000 claims abstract description 32
- 238000004809 thin layer chromatography Methods 0.000 claims abstract description 24
- 239000002904 solvent Substances 0.000 claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000003513 alkali Substances 0.000 claims abstract description 11
- 239000000463 material Substances 0.000 claims abstract description 7
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 51
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 42
- 241000218176 Corydalis Species 0.000 claims description 40
- 230000005526 G1 to G0 transition Effects 0.000 claims description 40
- 239000000523 sample Substances 0.000 claims description 34
- 239000007788 liquid Substances 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 19
- 230000002411 adverse Effects 0.000 claims description 15
- 239000002253 acid Substances 0.000 claims description 13
- 238000000605 extraction Methods 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- RHQQHZQUAMFINJ-GKWSUJDHSA-N 1-[(3s,5s,8s,9s,10s,11s,13s,14s,17s)-3,11-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-hydroxyethanone Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CC[C@H]21 RHQQHZQUAMFINJ-GKWSUJDHSA-N 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 11
- 238000010829 isocratic elution Methods 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 6
- 239000011707 mineral Substances 0.000 claims description 6
- 238000000967 suction filtration Methods 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 claims description 2
- 230000008021 deposition Effects 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 238000004811 liquid chromatography Methods 0.000 claims description 2
- 238000010298 pulverizing process Methods 0.000 claims description 2
- 238000002390 rotary evaporation Methods 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 abstract description 11
- 238000011084 recovery Methods 0.000 abstract description 10
- 239000003480 eluent Substances 0.000 abstract 2
- 238000004128 high performance liquid chromatography Methods 0.000 abstract 1
- 230000014759 maintenance of location Effects 0.000 abstract 1
- 150000007522 mineralic acids Chemical class 0.000 abstract 1
- 239000011259 mixed solution Substances 0.000 abstract 1
- 239000003795 chemical substances by application Substances 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 10
- 229940045641 monobasic sodium phosphate Drugs 0.000 description 7
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 7
- 238000005303 weighing Methods 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 240000003173 Drymaria cordata Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 241000543375 Sideroxylon Species 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Extraction Or Liquid Replacement (AREA)
Abstract
The invention discloses a method for separating dehydrocorydaline by pH-zone-refining countercurrent chromatography, which comprises the following steps of: (1) fully shaking chloroform, methanol and water, standing for demixing, adding inorganic acid of which the final concentration is 10 to 20nM into an upper phase serving as a fixed phase and adding alkali of which the final concentration is 10 to 20mM into a lower phase serving as a mobile phase; (2) dissolving the ethanol crude extract of yanhusuo in the fixed phase or mixed solution of the fixed phase and non-alkalified lower phase to prepare sample solution; and (3) filling the fixed phase into a separation column of a countercurrent chromatograph, pouring the sample solution to make the separation column rotate positively, continuously pouring the mobile phase at the flow rate of 1.5 to 2.5ml/min at the rotating speed of 750 to 850r/min, collecting eluent, detecting by thin layer chromatography and high performance liquid chromatography, combining the eluent of which the Rf value and retention time are the same as those of a dehydrocorydaline standard and the purity is over 90 percent, and recovering a solvent by using a rotary evaporator to obtain the dehydrocorydaline. The process is simple, recovery rate is high, the separated dehydrocorydaline has high purity, and the method plays an important role in the full utilization of the yanhusuo medicinal material.
Description
Technical field
The present invention relates to use the method for the refining adverse current chromatogram separating high-purity Dehydrocorydaline from the corydalis tuber crude extract of pH-district band.
Background technology
The act like a bully dry tuber of papaveracease (Popaveraceae) plant Yanhusuo of unit is a kind of in eight flavors of Zhejiang.In Traditional China medicine, Yanhusuo is considered to and can invigorates blood circulation, promoting the circulation of qi and alleviate all types of pain.The main active ingredient of corydalis tuber is a vegeto-alkali; Dehydrocorydaline is a kind of in the corydalis tuber quaternary ammonium hydroxide, and modern pharmacological research shows that Dehydrocorydaline can improve the hypoxia-bearing capability of mouse; Microcirculation improvement; The treatment myocardial ischemia, other there are some researches show that Dehydrocorydaline also can be used for treating ulcer and coronary heart disease.
At present, the separation of Dehydrocorydaline mainly contain column chromatography (Xiaoli Zhang, Qu Yang, Hou Jiaming, etc. the chemical ingredients of Yanhusuo [J], Shenyang Pharmaceutical University's journal, 2008,25 (7): 537-540), but separation efficiency is low and dead absorption arranged; Also useful preparative high performance liquid chromatography separate (Yang Jie, white snow, Xiao Haitao, etc. the preparation of Dehydrocorydaline reference substance and assay [J] in the Yanhusuo, the time precious traditional Chinese medical science traditional Chinese medicines, 2009,20 (4): 1000-1002), but applied sample amount is little and dead absorption arranged; Also have in addition the application standard high speed adverse current chromatogram separate (S. Q. Tong, J. Z, Yan, J. Z, Lou. J. Liq. Chromatogr. & Related Tech. 2005,28 (18): 2979-2989), but applied sample amount is too little.Band refining adverse current chromatogram (pH-zone-refining counter-current chromatography) in pH-district is the special adverse current chromatogram separation and preparation technology that on standard high speed adverse current chromatogram basis, grows up; Compare with the standard high speed adverse current chromatogram; Its outstanding characteristics are in stationary phase, to have added to keep acid (or alkali); In moving phase, added wash-out alkali (or acid), each component realizes separating according to its acid-basicity and hydrophobic difference in the sample, and target components is come out by wash-out with the rectangle peak; Impurity then is enriched in edge, rectangle peak, and different components is come out to be accompanied by the variation of pH value by wash-out.It is efficient that band refining adverse current chromatogram in pH-district has been inherited the standard high speed adverse current chromatogram, pollution-free to stationary phase, and the sample recovery rate advantages of higher has advantages such as the separation capacity is big, component is highly concentrated simultaneously.At present, band refining adverse current chromatogram in pH-district has been widely used in the separation of vegeto-alkali, peptide class and verivate, isomer etc.
Summary of the invention
The purpose of this invention is to provide the method that band refining adverse current chromatogram in a kind of pH-of application district separates Dehydrocorydaline, this method technology is simple, and separation efficiency is high.
The technical scheme that the present invention adopts is following:
A kind of method of using the refining adverse current chromatogram separation of pH-district band Dehydrocorydaline, described method may further comprise the steps:
(1) in separating funnel, adding chloroform, methyl alcohol, water respectively is the solvent system of 1.5～3:1～1.5:1 composition with volume ratio; Standing demix behind the shake well; Get be added to final concentration be the mineral acid of 10～20mM (preferred 10mM) as stationary phase, the alkali that is added to final concentration down and is 10～20mM (preferred 10mM) is as moving phase;
(2) the ethanol crude extract of getting corydalis tuber dissolves with stationary phase, and the ethanol crude extract that described stationary phase add-on is 25～60ml/g corydalis tuber makes sample solution after the dissolving;
(3) get the separator column that stationary phase is filled with counter current chromatograph, inject sample solution subsequently, treat that sample introduction is accomplished after; The opening speed unit just changes separator column, under 750～850r/min rotating speed; Flow velocity with 1.5～2.5ml/min continues to inject moving phase, with the UV-detector detection of wavelength 190～400nm, collects elutriant with automatic Fraction Collector; Thin-layer chromatography is followed the tracks of and to be detected no Dehydrocorydaline component to the elutriant, stops collection;
(4) detect through thin-layer chromatography and performance liquid collecting the elutriant that obtains in the step (3), merge and Dehydrocorydaline standard substance R
fValue and RT is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain Dehydrocorydaline.
In the said step (3), the volume of the sample introduction of said sample solution is usually less than 30% of separator column column volume.
In the said step (2), the following phased soln that the ethanol crude extract of corydalis tuber can also be used stationary phase and not alkalize, described method may further comprise the steps:
(1) in separating funnel, adding chloroform, methyl alcohol, water respectively is the solvent system of 1.5～3:1～1.5:1 composition with volume ratio; Standing demix behind the shake well; Get be added to final concentration be the mineral acid of 10～20mM as stationary phase, the alkali that is added to final concentration down and is 10～20mM is as moving phase;
(2) the ethanol crude extract of getting corydalis tuber is with the stationary phase and the following phased soln of alkalization not; The ethanol crude extract that described stationary phase add-on is 25～60ml/g corydalis tuber; The ethanol crude extract that the described not following amount of being added to of alkalization is 0～50ml/g corydalis tuber makes sample solution after the dissolving; 0 be meant not the following phase of alkalization add-on be infinitely close to 0 but be not 0;
(3) get the separator column that stationary phase is filled with counter current chromatograph, inject sample solution subsequently, treat that sample introduction is accomplished after; The opening speed unit just changes separator column, under 750～850r/min rotating speed; Flow velocity with 1.5～2.5ml/min continues to inject moving phase, with the UV-detector detection of wavelength 190～400nm, collects elutriant with automatic Fraction Collector; Thin-layer chromatography is followed the tracks of and to be detected no Dehydrocorydaline component to the elutriant, stops collection;
(4) detect through thin-layer chromatography and performance liquid collecting the elutriant that obtains in the step (3), merge and Dehydrocorydaline standard substance R
fValue and RT is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain Dehydrocorydaline.
The ethanol crude extract of described corydalis tuber makes as follows: get the corydalis tuber pulverizing medicinal materials; With 60～98% alcohol reflux 1～5 time; The extracting solution that merges each time, the suction filtration rear filtrate is evaporated to the medicinal extract shape, and using acid solution to pinch and dissolving to pinching the pH value of solution value is 2～3; Left standstill 10～30 hours, and got supernatant liquid filtering and remove deposition; Filtrating is collected the combined chloroform layer with chloroform extraction 1～5 time, and it is the extraction of 1～3% sodium hydroxide solution that the chloroform layer of merging uses mass concentration, gets the chloroform layer rotary evaporation and reclaims chloroform, gets the ethanol crude extract of corydalis tuber.Described acid solution is that mass concentration is 1～2% aqueous hydrochloric acid or 1～2% aqueous sulfuric acid.Said alcoholic acid volumetric usage is counted 10～20L/kg with the quality of corydalis tuber medicinal material usually.
The volume ratio of chloroform, first alcohol and water is preferably 2:1:1 in the solvent system described in the step according to the invention (1).
In the step according to the invention (1), described mineral acid is preferably hydrochloric acid or sulfuric acid, most preferably hydrochloric acid.
In the said step (1), described alkali is preferably triethylamine or diethylamine, most preferably is triethylamine.
In the said step (3), said injection sample solution can inject sample solution or pump into sample solution through pump through the sample introduction circle.
In the said step (4), the testing conditions of described performance liquid chromatography is: YMC ODS C
18Column (4.6 * 250mm, 5 μ m); Column temperature: 30 ℃; Moving phase: acetonitrile-0.03mol/L biphosphate sodium water solution (32:68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Detect wavelength: 345nm; Sample size: 20 μ l.
In said step (3) and (4), the developping agent of said thin-layer chromatography is that chloroform, methyl alcohol are with volume ratio 8:1 blended solvent.
Adopt half preparation type counter current chromatograph (column volume of separator column is 250ml) in the embodiment of the invention, counter current chromatograph is made up of constant flow pump, main frame (separator column), UV-detector, registering instrument etc.
In the said step (3), the preferred 810r/min of said rotating speed, flow rate of mobile phase is preferably 2ml/min.
Beneficial effect of the present invention is: separate Dehydrocorydaline through the refining adverse current chromatogram of pH-district band, technology is simple, and the recovery is high, and the Dehydrocorydaline purity that separation obtains is high, and making full use of of corydalis tuber medicinal material played an important role.
Embodiment
Come the present invention is further specified with specific embodiment below, but protection scope of the present invention is not limited thereto.
Embodiment 1
Get corydalis tuber medicinal material 1500g and pulverize the back,, merge 3 times extracting solution by with quadrat method extraction 3 times with 70% alcohol reflux of 22.5L 30 minutes; The suction filtration rear filtrate is evaporated to the medicinal extract shape, and using 2% hydrochloric acid soln to pinch and dissolving to pinching the pH value of solution value is 3, leaves standstill 15h; Suction filtration, filtrating are with 4 * 400mL chloroform extraction, and the combined chloroform layer also extracts with 2% sodium hydroxide solution; Get chloroform layer, reclaim chloroform, get corydalis tuber crude extract 10.440g with Rotary Evaporators.
Chloroform, first alcohol and water are disposed in the separating funnel according to volume ratio 2:1:1; Standing demix behind the shake well, on be added to hydrochloric acid, the final concentration that makes hydrochloric acid is that 10mM is as stationary phase; Under be added to triethylamine, the final concentration that makes triethylamine is that 10mM is as moving phase; Take by weighing corydalis tuber crude extract 208mg, the following phased soln with 8ml stationary phase and 8ml do not alkalize obtains sample solution.
Adopt half preparation type counter current chromatograph that the corydalis tuber crude extract is separated, the column volume of separator column is 250ml.At first stationary phase is filled with separator column with the flow velocity of 30ml/min, then above-mentioned sample solution is injected separator column through the sample introduction circle, treat that sample introduction is accomplished after; The opening speed unit just changes separator column, and the adjusting rotating speed is 810r/min; The flow velocity that moving phase is set is 2ml/min, begins to pump into moving phase, with the UV-detector detection of wavelength 254nm; Through chromatographic working station record color atlas and with the speed reception elutriant of automatic Fraction Collector by 5 minutes/test tube; (developping agent is chloroform: methyl alcohol=8:1, v/v) follows the tracks of and detects no Dehydrocorydaline component to the elutriant, stops collection with thin-layer chromatography.
(developping agent is chloroform: methyl alcohol=8:1 to the elutriant that collection obtains, and v/v) detects with performance liquid, merges and Dehydrocorydaline standard substance R through thin-layer chromatography
fValue (R
f=0.5) and RT (20.4min) is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the 22mg Dehydrocorydaline, purity is 98%, the recovery is 87%.
Performance liquid condition: YMC ODS C
18Post (4.6 * 250mm); Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32:68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Column temperature: 30 ℃; Detect wavelength: 345nm; Sample size: 20 μ l.
Embodiment 2
Get corydalis tuber medicinal material 2800g and pulverize the back,, merge 3 times extracting solution by with quadrat method extraction 3 times with 80% alcohol reflux of 42L 60 minutes; Be evaporated to the medicinal extract shape behind the suction filtration, using 2% hydrochloric acid soln to pinch and dissolving to pinching the pH value of solution value is 3, leaves standstill 20h; Suction filtration, filtrating are with 4 * 800mL chloroform extraction, and the combined chloroform layer also extracts with 2% sodium hydroxide solution; Get chloroform layer, reclaim chloroform, get corydalis tuber crude extract 17.1g with Rotary Evaporators.
Chloroform, first alcohol and water are disposed in the separating funnel according to volume ratio 1.5:1.5:1; Standing demix behind the shake well, on be added to hydrochloric acid, the final concentration that makes hydrochloric acid is that 10mM is as stationary phase; Under be added to triethylamine, the final concentration that makes triethylamine is that 10mM is as moving phase; Take by weighing corydalis tuber crude extract 205mg, the following phased soln with 8ml stationary phase and 8ml do not alkalize obtains sample solution.
Adopt half preparation type counter current chromatograph that the corydalis tuber crude extract is separated, the column volume of separator column is 250ml.At first stationary phase is filled with separator column with the flow velocity of 30ml/min, then above-mentioned sample solution is injected separator column through the sample introduction circle, treat that sample introduction is accomplished after; The opening speed unit just changes separator column, and the adjusting rotating speed is 800r/min; The flow velocity that moving phase is set is 2ml/min, begins to pump into moving phase, with the UV-detector detection of wavelength 254nm; Through chromatographic working station record color atlas and with the speed reception elutriant of automatic Fraction Collector by 5 minutes/test tube; (developping agent is chloroform: methyl alcohol=8:1, v/v) follows the tracks of and detects no Dehydrocorydaline component to the elutriant, stops collection with thin-layer chromatography.
(developping agent is chloroform: methyl alcohol=8:1 to the elutriant that collection obtains, and v/v) detects with performance liquid, merges and Dehydrocorydaline standard substance R through thin-layer chromatography
fValue (R
f=0.5) and RT (20.4min) is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the 11mg Dehydrocorydaline, purity is 97.4%, the recovery is 58.1%.
Performance liquid condition: YMC ODS C
18Post (4.6 * 250mm); Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32:68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Column temperature: 30 ℃; Detect wavelength: 345nm; Sample size: 20 μ l.
Embodiment 3
Chloroform, first alcohol and water are disposed in the separating funnel according to volume ratio 3:1.5:1; Standing demix behind the shake well, on be added to hydrochloric acid, the final concentration that makes hydrochloric acid is that 10mM is as stationary phase; Under be added to triethylamine, the final concentration that makes triethylamine is that 10mM is as moving phase; Take by weighing the corydalis tuber crude extract 205mg that extraction obtains among the embodiment 2, the following phased soln with 8ml stationary phase and 8ml do not alkalize obtains sample solution.
Adopt half preparation type counter current chromatograph that the corydalis tuber crude extract is separated, the column volume of separator column is 250ml.At first stationary phase is filled with separator column with the flow velocity of 30ml/min, then above-mentioned sample solution is injected separator column through the sample introduction circle, treat that sample introduction is accomplished after; The opening speed unit just changes separator column, and the adjusting rotating speed is 810r/min; The flow velocity that moving phase is set is 2ml/min, begins to pump into moving phase, with the UV-detector detection of wavelength 254nm; Through chromatographic working station record color atlas and with the speed reception elutriant of automatic Fraction Collector by 5 minutes/test tube; (developping agent is chloroform: methyl alcohol=8:1, v/v) follows the tracks of and detects no Dehydrocorydaline component to the elutriant, stops collection with thin-layer chromatography.
(developping agent is chloroform: methyl alcohol=8:1 to the elutriant that collection obtains, and v/v) detects with performance liquid, merges and Dehydrocorydaline standard substance R through thin-layer chromatography
fValue (R
f=0.5) and RT (20.4min) is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the 15mg Dehydrocorydaline, purity is 98.1%, the recovery is 79.8%.
Performance liquid condition: YMC ODS C
18Post (4.6 * 250mm); Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32:68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Column temperature: 30 ℃; Detect wavelength: 345nm; Sample size: 20 μ l.
Embodiment 4
Chloroform, first alcohol and water are disposed in the separating funnel according to volume ratio 3:1:1; Standing demix behind the shake well, on be added to hydrochloric acid, the final concentration that makes hydrochloric acid is that 10mM is as stationary phase; Under be added to triethylamine, the final concentration that makes triethylamine is that 10mM is as moving phase; Take by weighing the corydalis tuber crude extract 222mg that extraction obtains among the embodiment 2, the following phased soln with 8ml stationary phase and 8ml do not alkalize obtains sample solution.
Adopt half preparation type counter current chromatograph that the corydalis tuber crude extract is separated, the column volume of separator column is 250ml.At first stationary phase is filled with separator column with the flow velocity of 30ml/min, then above-mentioned sample solution is injected separator column through the sample introduction circle, treat that sample introduction is accomplished after; The opening speed unit just changes separator column, and the adjusting rotating speed is 810r/min; The flow velocity that moving phase is set is 2ml/min, begins to pump into moving phase, with the UV-detector detection of wavelength 254nm; Through chromatographic working station record color atlas and with the speed reception elutriant of automatic Fraction Collector by 5 minutes/test tube; (developping agent is chloroform: methyl alcohol=8:1, v/v) follows the tracks of and detects no Dehydrocorydaline component to the elutriant, stops collection with thin-layer chromatography.
(developping agent is chloroform: methyl alcohol=8:1 to the elutriant that collection obtains, and v/v) detects with performance liquid, merges and Dehydrocorydaline standard substance R through thin-layer chromatography
fValue (R
f=0.5) and RT (20.4min) is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the 15mg Dehydrocorydaline, purity is 99.3%, the recovery is 74.6%.
Performance liquid condition: YMC ODS C
18Post (4.6 * 250mm); Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32:68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Column temperature: 30 ℃; Detect wavelength: 345nm; Sample size: 20 μ l.
Embodiment 5
Chloroform, first alcohol and water are disposed in the separating funnel according to volume ratio 1.5:1:1; Standing demix behind the shake well, on be added to hydrochloric acid, the final concentration that makes hydrochloric acid is that 10mM is as stationary phase; Under be added to triethylamine, the final concentration that makes triethylamine is that 10mM is as moving phase; Take by weighing the corydalis tuber crude extract 205mg that extraction obtains among the embodiment 2, the following phased soln with 8ml stationary phase and 8ml do not alkalize obtains sample solution.
Adopt half preparation type counter current chromatograph that the corydalis tuber crude extract is separated, the column volume of separator column is 250ml.At first stationary phase is filled with separator column with the flow velocity of 30ml/min, then above-mentioned sample solution is injected separator column through the sample introduction circle, treat that sample introduction is accomplished after; The opening speed unit just changes separator column, and the adjusting rotating speed is 810r/min; The flow velocity that moving phase is set is 2ml/min, begins to pump into moving phase, with the UV-detector detection of wavelength 254nm; Through chromatographic working station record color atlas and with the speed reception elutriant of automatic Fraction Collector by 5 minutes/test tube; (developping agent is chloroform: methyl alcohol=8:1, v/v) follows the tracks of and detects no Dehydrocorydaline component to the elutriant, stops collection with thin-layer chromatography.
(developping agent is chloroform: methyl alcohol=8:1 to the elutriant that collection obtains, and v/v) detects with performance liquid, merges and Dehydrocorydaline standard substance R through thin-layer chromatography
fValue (R
f=0.5) and RT (20.4min) is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the 12mg Dehydrocorydaline, purity is 99.5%, the recovery is 64.8%.
Performance liquid condition: YMC ODS C
18Post (4.6 * 250mm); Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32:68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Column temperature: 30 ℃; Detect wavelength: 345nm; Sample size: 20 μ l.
Embodiment 6
Chloroform, first alcohol and water are disposed in the separating funnel according to volume ratio 2:1:1; Standing demix behind the shake well, on be added to hydrochloric acid, the final concentration that makes hydrochloric acid is that 20mM is as stationary phase; Under be added to triethylamine, the final concentration that makes triethylamine is that 20mM is as moving phase; Take by weighing the corydalis tuber crude extract 222mg that extraction obtains among the embodiment 2,, obtain sample solution with the dissolving of 13.3ml stationary phase.
Adopt half preparation type counter current chromatograph that the corydalis tuber crude extract is separated, the column volume of separator column is 250ml.At first stationary phase is filled with separator column with the flow velocity of 30ml/min, then above-mentioned sample solution is injected separator column through pump, treat that sample introduction is accomplished after; The opening speed unit just changes separator column, and the adjusting rotating speed is 850r/min; The flow velocity that moving phase is set is 2.5ml/min, begins to pump into moving phase, with the UV-detector detection of wavelength 254nm; Through chromatographic working station record color atlas and with the speed reception elutriant of automatic Fraction Collector by 4 minutes/test tube; (developping agent is chloroform: methyl alcohol=8:1, v/v) follows the tracks of and detects no Dehydrocorydaline component to the elutriant, stops collection with thin-layer chromatography.
(developping agent is chloroform: methyl alcohol=8:1 to the elutriant that collection obtains, and v/v) detects with performance liquid, merges and Dehydrocorydaline standard substance R through thin-layer chromatography
fValue (R
f=0.5) and RT (20.4min) is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the 18mg Dehydrocorydaline, purity is 94.4%, the recovery is 85.1%.
Performance liquid condition: YMC ODS C
18Post (4.6 * 250mm); Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32:68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Column temperature: 30 ℃; Detect wavelength: 345nm; Sample size: 20 μ l.
Embodiment 7
Chloroform, first alcohol and water are disposed in the separating funnel according to volume ratio 2:1:1; Standing demix behind the shake well, on be added to hydrochloric acid, the final concentration that makes hydrochloric acid is that 10mM is as stationary phase; Under be added to triethylamine, the final concentration that makes triethylamine is that 10mM is as moving phase; Take by weighing the corydalis tuber crude extract 200mg that extraction obtains among the embodiment 2, the following phased soln with 5ml stationary phase and 10ml do not alkalize obtains sample solution.
Adopt half preparation type counter current chromatograph that the corydalis tuber crude extract is separated, the column volume of separator column is 250ml.At first stationary phase is filled with separator column with the flow velocity of 30ml/min, then above-mentioned sample solution is injected separator column through the sample introduction circle, treat that sample introduction is accomplished after; The opening speed unit just changes separator column, and the adjusting rotating speed is 750r/min; The flow velocity that moving phase is set is 1.5ml/min, begins to pump into moving phase, with the UV-detector detection of wavelength 254nm; Through chromatographic working station record color atlas and with the speed reception elutriant of automatic Fraction Collector by 5 minutes/test tube; (developping agent is chloroform: methyl alcohol=8:1, v/v) follows the tracks of and detects no Dehydrocorydaline component to the elutriant, stops collection with thin-layer chromatography.
(developping agent is chloroform: methyl alcohol=8:1 to the elutriant that collection obtains, and v/v) detects with performance liquid, merges and Dehydrocorydaline standard substance R through thin-layer chromatography
fValue (R
f=0.5) and RT (20.4min) is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the 16mg Dehydrocorydaline, purity is 95.3%, the recovery is 84.8%.
Performance liquid condition: YMC ODS C
18Post (4.6 * 250mm); Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32:68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Column temperature: 30 ℃; Detect wavelength: 345nm; Sample size: 20 μ l.
Claims (9)
1. use the method that band refining adverse current chromatogram in pH-district separates Dehydrocorydaline for one kind, it is characterized in that described method may further comprise the steps:
(1) in separating funnel, adding chloroform, methyl alcohol, water respectively is 1.5～3: 1～1.5 with volume ratio: 1 solvent system formed; Standing demix behind the shake well; Get be added to final concentration be the mineral acid of 10～20mM as stationary phase, the alkali that is added to final concentration down and is 10～20mM is as moving phase;
(2) the ethanol crude extract of getting corydalis tuber dissolves with stationary phase, and the ethanol crude extract that described stationary phase add-on is 25～60ml/g corydalis tuber is processed sample solution after the dissolving; The ethanol crude extract of described corydalis tuber makes as follows: get the corydalis tuber pulverizing medicinal materials; With 60～98% alcohol reflux 1～5 time; The extracting solution that merges each time, the suction filtration rear filtrate is evaporated to the medicinal extract shape, use acid solution to pinch to dissolve to the pH value of pinching solution be 2～3; Left standstill 10～30 hours, and got supernatant liquid filtering and remove deposition; Filtrating is collected the combined chloroform layer with chloroform extraction 1～5 time, and it is the extraction of 1～3% sodium hydroxide solution that the chloroform layer of merging uses mass concentration, gets the chloroform layer rotary evaporation and reclaims chloroform, gets the ethanol crude extract of corydalis tuber;
(3) get the separator column that stationary phase is filled with counter current chromatograph, inject sample solution subsequently, treat that sample introduction is accomplished after; The opening speed unit just changes separator column, under 750～850r/min rotating speed; Flow velocity with 1.5～2.5ml/min continues to inject moving phase, with the UV-detector detection of wavelength 190～400nm, collects elutriant with automatic Fraction Collector; Thin-layer chromatography is followed the tracks of and to be detected no Dehydrocorydaline component to the elutriant, stops collection;
(4) detect through thin-layer chromatography and performance liquid collecting the elutriant that obtains in the step (3), merge and Dehydrocorydaline standard substance R
fValue and RT is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain Dehydrocorydaline.
2. the method that the refining adverse current chromatogram of application pH-as claimed in claim 1 district's band separates Dehydrocorydaline is characterized in that described method may further comprise the steps:
(1) in separating funnel, adding chloroform, methyl alcohol, water respectively is 1.5～3: 1～1.5 with volume ratio: 1 solvent system formed; Standing demix behind the shake well; Get be added to final concentration be the mineral acid of 10～20mM as stationary phase, the alkali that is added to final concentration down and is 10～20mM is as moving phase;
(2) the ethanol crude extract of getting corydalis tuber is with the stationary phase and the following phased soln of alkalization not; The ethanol crude extract that described stationary phase add-on is 25～60ml/g corydalis tuber; The ethanol crude extract that the described not following amount of being added to of alkalization is 0～50ml/g corydalis tuber is processed sample solution after the dissolving;
(3) get the separator column that stationary phase is filled with counter current chromatograph, inject sample solution subsequently, treat that sample introduction is accomplished after; The opening speed unit just changes separator column, under 750～850r/min rotating speed; Flow velocity with 1.5～2.5ml/min continues to inject moving phase, with the UV-detector detection of wavelength 190～400nm, collects elutriant with automatic Fraction Collector; TLC follows the tracks of and to detect no Dehydrocorydaline component to the elutriant, stops collection;
(4) detect through thin-layer chromatography and performance liquid collecting the elutriant that obtains in the step (3), merge and Dehydrocorydaline standard substance R
fValue and RT is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain Dehydrocorydaline.
3. the method for claim 1 is characterized in that described acid solution is that mass concentration is 1～2% aqueous hydrochloric acid or 1～2% aqueous sulfuric acid.
4. according to claim 1 or claim 2 method is characterized in that the volume of the sample introduction of said sample solution is less than 30% of the separator column column volume in the said step (3).
5. according to claim 1 or claim 2 method, the volume ratio that it is characterized in that chloroform, first alcohol and water in the solvent system described in the said step (1) is 2: 1: 1.
6. according to claim 1 or claim 2 method is characterized in that in the said step (1), described mineral acid is a hydrochloric acid.
7. according to claim 1 or claim 2 method is characterized in that in the said step (1), described alkali is triethylamine.
8. according to claim 1 or claim 2 method is characterized in that in the said step (3), and said injection sample solution can inject sample solution or pump into sample solution through pump through the sample introduction circle.
9. according to claim 1 or claim 2 method is characterized in that the testing conditions of the described performance liquid chromatography of step (4) is: YMC ODS C
18Column 4.6 * 250mm, 5 μ m; Column temperature: 30 ℃; Moving phase: acetonitrile-0.03mol/L biphosphate sodium water solution is with 32: 68 blended solution of volume ratio, isocratic elution; Flow velocity: 0.6ml/min; Detect wavelength: 345nm; Sample size: 20 μ l.
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